Vitamin D3/VDR inhibits inflammation through NF-κB pathway accompanied by resisting apoptosis and inducing autophagy in abalone Haliotis discus hannai.

Vitamin D3 is believed to be a contributing factor to innate immunity. Vitamin D receptor (VDR) has a positive effect on inhibiting nuclear factor κB (NF-κB)-mediated inflammation. The underlying molecular mechanisms remain unclear, particularly in mollusks. Consequently, this study will investigate the process of vitamin D3/VDR regulating NF-κB pathway and further explore their functions on inflammation, autophagy, and apoptosis in abalone Haliotis discus hannai. Results showed that knockdown of VDR by using siRNA and dsRNA of VDR in vitro and in vivo led to more intense response of NF-κB signaling to lipopolysaccharide and higher level of apoptosis and autophagy. In addition, 1,25(OH)2D3 stimulation after VDR silencing could partially alleviate apoptosis and induce autophagy. Overexpression of VDR restricted the K48-polyubiquitin chain-dependent inhibitor of κB (IκB) ubiquitination and apoptosis-associated speck-like protein containing CARD (ASC) oligomerization. Besides, VDR silencing resulted in increase of ASC speck formation. In further mechanistic studies, we showed that VDR can directly bind to IκB and IKK1 in vitro and in vivo. In the feeding trial, H&E staining, TUNEL, and electron microscope results showed that vitamin D3 deficiency (0 IU/kg) could recruit more basophilic cells and increase more TUNEL-positive apoptotic cells and lipid droplets (LDs) than vitamin D3 supplement (1000 IU/kg and 5000 IU/kg). In summary, abalone VDR plays a negative regulator role in NF-κB-mediated inflammation via interacting with IκB and inhibiting ubiquitin-dependent degradation of IκB. Vitamin D3 in combination with VDR is essential to establish a delicate balance between autophagy and apoptosis in response to inflammation.

Biotin alleviates hepatic and intestinal inflammation and apoptosis induced by high dietary carbohydrate in juvenile turbot (Scophthalmus maximus L.).

Excessive dietary carbohydrate commonly impairs the functions of liver and intestine in carnivorous fish. In the present study, a 10-week feeding trial was carried out to explore the regulation of biotin on the hepatic and intestinal inflammation and apoptosis in turbot (Scophthalmus maximus L.) fed with high carbohydrate diets. Three isonitrogenous and isolipidic experimental diets were designed as follows: the CC diet with 18.6% of carbohydrate and 0.04 mg/kg of biotin, the HC diet with 26.9% of carbohydrate and 0.05 mg/kg of biotin, and the HCB diet with 26.9% of carbohydrate and 1.62 mg/kg of biotin. Results showed that high dietary carbohydrate (HC diet) impaired the morphology of liver and intestine, however, inclusion of dietary biotin (HCB diet) normalized their morphology. Inflammation-related gene expression of nuclear factor κB p65 (nf-κb p65), tumor necrosis factor α (tnf-α), interleukin-1ß (il-1ß), il-6 and il-8, and the protein expression of NF-κB p65 in the liver and intestine were significantly up-regulated in the HC group compared to those in the CC group (P < 0.05), the HCB diet decreased their expression compared to the HC group (P < 0.05). The gene expression of il-10 and transforming growth factor-ß (tgf-ß) in the liver and intestine were significantly decreased in the HC group compared to the CC group (P < 0.05), and inclusion of dietary biotin increased the il-10 and tgf-ß expression in the liver and intestine (P < 0.05). Moreover, compared to the CC group, the HC group had a stronger degree of DNA fragmentation and more TUNEL-positive cells in the liver and intestine, and the HCB group had a slighter degree of DNA fragmentation and fewer TUNEL-positive cells compared to the HC group. Meanwhile, the gene expression of B-cell lymphoma protein-2-associated X protein (bax) and executor apoptosis-related cysteine peptidase 3 (caspase-3) were significantly up-regulated and the gene expression of B-cell lymphoma-2 (bcl-2) was significantly down-regulated both in the liver and intestine in the HC group compared with those in the CC group (P < 0.05). Inclusion of dietary biotin significantly decreased the bax and caspase-3 mRNA levels and increased bcl-2 mRNA level in the liver and intestine (P < 0.05). In conclusion, high dietary carbohydrate (26.9% vs 18.6%) induced inflammation and apoptosis in liver and intestine. Supplementation of biotin (1.62 mg/kg vs 0.05 mg/kg) in diet can alleviate the high-dietary-carbohydrate-induced hepatic and intestinal inflammation as well as inhibit apoptosis in turbot. The present study provides basic data for the application of biotin into feed, especially the high-carbohydrate feed for turbot.

Arginine Regulates TOR Signaling Pathway through SLC38A9 in Abalone Haliotis discus hannai.

Arginine plays an important role in the regulation of the target of the rapamycin (TOR) signaling pathway, and Solute Carrier Family 38 Member 9 (SLC38A9) was identified to participate in the amino acid-dependent activation of TOR in humans. However, the regulations of arginine on the TOR signaling pathway in abalone are still unclear. In this study, slc38a9 of abalone was cloned, and the slc38a9 was knocked down and overexpressed to explore its function in the regulation of the TOR signaling pathway. The results showed that knockdown of slc38a9 decreased the expression of tor, ribosomal s6 protein kinase (s6k) and eukaryotic translation initiation factor 4e (eif4e) and inhibited the activation of the TOR signaling pathway by arginine. Overexpression of slc38a9 up-regulated the expression of TOR-related genes. In addition, hemocytes of abalone were treated with 0, 0.2, 0.5, 1, 2 and 4 mmol/L of arginine, and abalones were fed diets with 1.17%, 1.68% and 3.43% of arginine, respectively, for 120 days. Supplementation of arginine (0.5-4 mmol/L) increased the expressions of slc38a9, tor, s6k and eif4e in hemocytes, and abalone fed with 1.68% of dietary arginine showed higher mRNA levels of slc38a9, tor, s6k and eif4e and phosphorylation levels of TOR, S6 and 4E-BP. In conclusion, the TOR signaling pathway of abalone can be regulated by arginine, and SLC38A9 plays an essential role in this regulation.

Ascorbic Acid Regulates the Immunity, Anti-Oxidation and Apoptosis in Abalone Haliotis discus hannai Ino.

The present study was conducted to investigate the roles of ascorbic acid (AA) in immune response, anti-oxidation and apoptosis in abalone (Haliotis discus hannai Ino). Seven semi-purified diets with graded levels of AA (0, 50, 100, 200, 500, 1000 and 5000 mg/kg) were fed to abalone (initial weight: 12.01 ± 0.001 g, initial shell length: 48.44 ± 0.069 mm) for 100 days. The survival, weight gain rate and daily increment in shell length were not affected by dietary AA. The AA content in the gill, muscle and digestive glands of abalone was significantly increased by dietary AA. In terms of immunity, dietary AA significantly improved the total hemocyte count, respiratory burst and phagocytic activity in hemolymph, and lysozyme activity in cell-free hemolymph (CFH). In the digestive gland, the TLR-MyD88-dependent and TLR-MyD88-independent signaling pathways were suppressed by dietary AA supplementation. The mRNA levels of ß-defensin and arginase-I in the digestive gland were significantly increased by dietary AA. In the gill, only the TLR-MyD88-dependent signaling pathway was depressed by dietary AA to reduce inflammation in abalone. The level of mytimacin 6 in the gill was significantly upregulated by dietary AA. After Vibrio parahaemolyticus infection, the TLR signaling pathway in the digestive gland was suppressed by dietary AA, which reduced inflammation in the abalone. In terms of anti-oxidation, superoxide dismutase, glutathione peroxidase and catalase activities, as well as total anti-oxidative capacity and reduced glutathione content in CFH, were all significantly upregulated. The malondialdehyde content was significantly downregulated by dietary AA. The anti-oxidative capacity was improved by triggering the Keap1-Nrf2 pathway in abalone. In terms of apoptosis, dietary AA could enhance the anti-apoptosis ability via the JNK-Bcl-2/Bax signaling cascade in abalone. To conclude, dietary AA was involved in regulating immunity, anti-oxidation and apoptosis in abalone.

Dietary taurine modulates hepatic oxidative status, ER stress and inflammation in juvenile turbot (Scophthalmus maximus L.) fed high carbohydrate diets.

This study was conducted to explore the beneficial role of taurine against chronic high carbohydrate diet-induced oxidative stress, endoplasmic reticulum (ER) stress and inflammation, and to understand the underlying molecular mechanisms in turbot. Two 10-week feeding trials were simultaneously conducted. For the one, six experimental diets with graded levels of taurine supplementation (0, 0.4%, 0.8%, 1.2%, 1.6% and, 2.0%, respectively) and 15% of carbohydrate were used. For the other one, three graded levels of dietary taurine supplementation (0.4%, 1.2% and 2.0%, respectively) with 21% of carbohydrate were used. The results showed that higher expression level of inflammation cytokines and ER stress related genes were detected in higher dietary carbohydrate group. In both feeding trials, 1.2% of dietary taurine supplementation improved anti-oxidative status by decreasing the content of malondialdehyde, increasing the catalase activity and total anti-oxidative capacities. In feeding trial 1, appropriate taurine supplementation lowered contents of tumour necrosis factor-a, interleukin-6, aspartate aminotransferase and alkaline phosphatase in plasma, and decreased the expressions of pro-inflammatory cytokines, such as interleukin-8 (il-8) and interferon-γ (ifn-γ). Furthermore, dietary taurine reduced ER stress by decreasing the mRNA levels of activating transcription factor 6, protein kinase R-like endoplasmic reticulum kinase and G protein-coupled receptor 78. The optimal dietary taurine content was estimated as 1.40% based on the analysis of specific growth rate. In feeding trial 2, dietary taurine supplementation attenuated liver inflammation partly referring to significantly down-regulated mRNA levels of nuclear transcription factor-κB p65, ifn-γ, interleukin1ß and up-regulate the transcript of ribosomal protein S6 kinase 1. Dietary taurine supplementation in feeding trial 2 significantly increased the Nrf2-related factor 2 protein level and decreased the NFκB p65 protein level only at 21% of dietary carbohydrate level. Taurine can alleviate the oxidative damage and inflammation caused by 21% of dietary carbohydrate to a certain degree. Overall, the present study confirmed that dietary taurine supplementation improved growth performance and anti-oxidative response, and reduced liver inflammatory and ER stress processes induced by high dietary carbohydrate in turbot.